Effect of CCAR2 depletion on the gene expression profile of U2OS cells

CCAR2 is a nuclear protein recently emerged as a pivotal player of the DNA damage response since it has been found involved in both apoptosis induction and DNA repair. Differently, its role in tumorigenesis and cancer progression is still elusive. In our studies we found that CCAR2 depletion impairs the proliferation of human cancer cell lines, but leaves unaffected the growth of normal immortalized cells. To better investigate this point we performed a genome wide gene expression analyses in U2OS and BJ-hTERT depleted of CCAR2 and we found that loss of this protein causes the deregulation of genes implicated in the AKT pathway specifically in U2OS cells, but not in BJ-hTERT. In accordance with these results we found a reduction in AKT activation in all the tested cancer cell lines depleted of CCAR2, but not in the normal ones. The defective activation of AKT is caused by the upregulation of TRB3 gene in cancer cells depleted of CCAR2 and finally results in the reduction of GSK3β phosphorylation, prevention of G1/S transition and inhibition of cancer cell growth. The human osteosarcoma cell line U2OS was cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% of fetal calf serum. Cells were transfected with control or a pool of 4 specific RIBOXX siRNAs against CCAR2 using RNAiMAX (Thermo Fisher) according to manufacturer proteocols. 6 days after transfection cells were harvested in QIAzol (Qiagen) and the extracted RNA was used for gene expression analyses. Samples were prepared in indipendent triplicates.

CCAR2是一种核蛋白，近年被确立为DNA损伤应答的核心调控因子，因其被证实同时参与细胞凋亡诱导与DNA修复过程。与之不同的是，其在肿瘤发生与癌症进展中的作用仍不明确。本研究团队发现，敲低CCAR2会削弱人类癌细胞系的增殖能力，但对永生化正常细胞的生长无显著影响。为深入探究这一现象，我们对CCAR2敲低的U2OS细胞与BJ-hTERT细胞开展了全基因组基因表达分析，结果显示，该蛋白的缺失仅会导致U2OS细胞中AKT信号通路相关基因的表达失调，而BJ-hTERT细胞中并无此变化。与此一致的是，我们在所有被检测的CCAR2敲低癌细胞系中均观察到AKT激活水平降低，但正常细胞中未出现该现象。AKT激活缺陷的成因是CCAR2敲低的癌细胞中TRB3基因的表达上调，最终导致GSK3β磷酸化水平降低、G1/S期转换受阻，进而抑制癌细胞增殖。本研究中人骨肉瘤细胞系U2OS培养于添加10%胎牛血清的Dulbecco改良Eagle培养基（DMEM）中，使用RNAiMAX（Thermo Fisher）转染试剂，按照制造商的实验规程，将对照siRNA或靶向CCAR2的4条特异性RIBOXX小干扰RNA（siRNAs）混合池转染至细胞中，转染6天后，使用QIAzol（Qiagen）裂解收集细胞，提取的RNA用于基因表达分析，所有样本均设置独立的三次生物学重复。